Analysis of mutant huntingtin (htt) polyglutamine (polyQ) aggregation has suggested a role for this process in the pathogenesis of Huntington's Disease (HD). Biochemical information on mutant polyglutamine is incomplete, but commonalities with other neurodegenerative protein aggregation diseases suggest common features of neurodegenerative pathogenesis. While inclusion bodies themselves appear not to be the toxic species, aspects of protein conformational change and aggregation appear relevant to pathogenesis. To investigate biochemical features of mutant htt,we prepared a structure-based cell culture model of htt polyQ aggregation and toxicity by expressing htt exon-1 N-terminal fragment with a modified expanded polyQ region in mammalian cell lines and in cultured murine primary cortical neurons. The data show a correlation between htt polyQ proteins predicted to assume a compact beta-structure and toxicity. We have also developed an in vitro system for the biochemical and biophysical analysis of htt mutant polyQ aggregation, allowing us to probe the htt aggregation pathway. Globular and protofibrillar species were observed at early stages of aggregation, and may represent intermediates in the fibrillization process. In specific aim 1 of this proposal, we will use our cell culture model to characterizethe beta-sheet structural organization within htt mutant polyQ aggregates. In aim 2, we will use our in vitro model to investigate the relevance of the putative compact beta-sheet on htt mutant polyQ fibrillization. In aim 3, we will characterize the role of mutant htt intramolecular structure in polyQ-induced toxicity in mammalian cell culture. These studies will elucidate the relationship between mutant htt polyQ structure and neurotoxicity in HD.